JPH01262794A - Fibrous or filmy carrier for immobilized enzyme - Google Patents

Abstract

PURPOSE:To obtain a fibrous or filmy carrier for immobilized enzyme resistant to water-treatment, by irradiating a fibrous or filmy material with low- temperature plasma, graft-polymerizing a copolymerizing monomer to the surface of the material and immobilizing an enzyme by ion bonding method. CONSTITUTION:Preferably an organic or inorganic fibrous or filmy material is irradiated with low-temperature plasma using a plasma gas of nitrogen, argon, etc., and a copolymerizing monomer is graft-polymerized to the activated surface of the material. The objective carrier is produced by immobilizing an enzyme such as glucose oxidase or invertase to the graft-polymer by ion bonding method. The copolymerizing monomer is preferably a compound of formula I or formula II (R1, R2, R3 and R4 are H or alkyl; n is positive number). The above low-temperature plasma irradiation is carried out preferably by low-temperature plasma treatment with an inorganic gas having a gas pressure of 0.01-10Torr.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a fiber or film-like carrier for immobilized enzymes. More specifically, by subjecting the film material to low-temperature plasma treatment, the primary surface region is activated (radicalized), the copolymerized monomer is graft-polymerized, and then the grafted polymer and the enzyme are ionized. The present invention provides a novel fiber or film-like carrier for immobilized enzymes, which is immobilized by a bonding method.

It is known that L1-transferring enzymes generally proceed with the reaction under mild conditions such as room temperature, normal pressure, and near neutrality, and that the reaction has high substrate specificity. Currently, enzymes are mainly used in the food industry, but growth is also expected in the fermentation and chemical industries.

The industrial use of enzymes as catalysts has been practiced for a long time, mainly in the fermentation industry. However, in the past, enzymes were dissolved or dispersed in an aqueous solution and used for reactions, and the enzymes were not recovered from the reaction materials and were discarded after each reaction.

However, there has been remarkable research into enzyme immobilization technology that allows enzymes to be used repeatedly and stably.

Various methods are known for immobilizing such enzymes. These can be broadly classified into carrier bonding methods, crosslinking methods, and inclusion methods, and carrier bonding methods include covalent bonding methods, ionic bonding methods, and physical adsorption methods. The present invention is based on an ion bonding method, and ion exchange resins are widely known as carriers for this method.

Ion exchange resins having ion exchange ability in the form of styrene-divinylbenzene copolymers are difficult to process in water due to swelling and washing-off of the resin.

On the other hand, a fibrous carrier is characterized by its morphology, and its large surface area accelerates the exchange rate during enzyme binding, which is also advantageous for enzyme reactions. It also makes it easier to separate the reaction solution and product solution.
It can be used in any form such as non-woven fabric, woven fabric, thread-like, cotton-like, etc.

Many research examples using organic fibers as carriers for immobilized enzymes have been known. As a method of imparting ion exchange ability to organic fibers, there are two methods: one using a compound that has ionic properties in the 14th stage of the fiber material, and the other using a chemical reaction in a male form. As is known, fibers with carbon fibers are extremely resistant to water treatment and chemicals, but on the other hand, they are poor in chemical reactions, so it has been difficult to impart ion exchange ability to them.

■ ・1 The present inventors focused on the above points and further focused on the future potential of industrial immobilized enzymes, and as a result of conducting intensive and extensive systematic research on fiber and film-like carriers for immobilized enzymes, we found that:
We have completed the present invention by discovering that the desired effect can be obtained by irradiating fibers and film-like materials with low-temperature plasma, grafting copolymer monomers onto the surface of the materials, and then immobilizing enzymes.

An object of the present invention is to provide an enzyme-immobilized carrier made of fiber or film material that is robust against water treatment.

Another object is to provide a biosensor that detects chemical substances by using these immobilized enzyme carriers.

"L Rise 1" The fiber and film-like materials of the present invention mean organic or inorganic fibers and films thereof. Although fibers with larger surface areas are more desirable, films can also be effective in immobilizing enzymes. Further, the shape of the material introduced into the reaction vessel may be TOW-like, fibrous-like, short-fiber-like, non-woven fabric, film, woven fabric, thread-like, sliver-like, etc., all of which are applicable to the present invention.

The fibers and film-like materials of the present invention are irradiated with low-temperature plasma,
The determining factors of the plasma processing conditions for activating the surface region of the material are the gas composition, pressure, and flow rate, as well as the power and processing time, which determine the possibility of graft polymerization.

The plasma gas of the present invention includes nitrogen, oxygen, hydrogen, argon,
neon, helium, air, water vapor,.

There are chlorine, ammonia, carbon oxide, carbon dioxide, nitrous oxide, nitrogen dioxide, sulfur dioxide, etc., and there is also a polymerizable plasma gas, which can be used alone or in combination.

In order to achieve the objects of the present invention, it is desirable to create an atmosphere in which the partial pressure of the plasma gas is 1 torr or less, more preferably 10 -1 torr or less. In a plasma atmosphere with a partial pressure greater than 1 Torr, the effectiveness of plasma processing decreases rapidly. The flow rate of the plasma gas is determined by the volume of the reactor and the partial pressure of the plasma gas.

Although the output is generally 200 watts or less, it is possible to obtain the desired performance by combining it with the processing time.

When applying plasma gas to the surface of fibers or film materials, many combinations are possible.

That is, various choices can be made, such as the structure of the reactor, the type of power source, frequency, discharge format, and electrode position.

For plasma processing, the power source is high frequency +13.5
6MHz), microwave (2,450Hzl, low frequency (
(several KHz). Glow discharge is effective as a discharge method. Regarding the position of the electrode, there are internal and external types, but the external type is easier to operate when considering the next graft polymerization process.

Graft polymerization in the present invention means grafting a copolymer monomer onto the surface of fibers or film-form elements tS that have been activated (radicalized) by plasma treatment, and after plasma treatment, they are taken out into the atmosphere. However, it is preferable to carry out the graft polymerization in a vacuum state.

The copolymerizable monomer of the present invention means an ionic monomer represented by the chemical formula (r) or (I1) described in claim 3.

Examples of enzymes that can be immobilized by this invention include glucose oxidase, alcohol oxidase, invertase, glucose isomerase, lipase, and urease, and two or more of these may be used simultaneously.

[i-1] The feature of the present invention is to surface activate polyester-based, polyether-based fibers and film materials with low reactivity through low-temperature plasma treatment, and graft a copolymerizable monomer that ionically bonds with enzymes into the material. The object of the present invention is to provide a novel carrier for immobilized enzymes in which a reactive group can be easily introduced and a carrier bonding method can be applied.

Furthermore, it has many features such as strong bonding strength between fibers and enzymes, high sustainability and activity, and high efficiency of enzyme immobilization, and a large surface area that improves enzyme reaction efficiency.

Hereinafter, the present invention will be explained in more detail with reference to Examples.

In addition, the grafting rate and activity retention rate in Examples were measured by the following method.

(a) Grafting ratio The grafting ratio was determined from the difference between the dry weight of the graft-polymerized fibers after removing the homopolymer and the like by 7% washing and the dry electricity amount before the grafting treatment.

Weight before treatment: Activity retention The enzyme-immobilized fibers were treated with an aqueous hydrochloric acid solution, thoroughly washed with water, the enzyme activity at the first measurement was taken as A1, and then the ## immobilized fibers were placed in 1 MNFrl for 30 days. The enzyme activity after soaking was defined as A2, and the ratio was determined.

Activity retention rate (%) = A2/AI x 100 Example 1 Polyester fiber (I
'OOX 200 IIm) 1 g φ100
The tube was suspended in a glass chamber of X200, and the chamber was sufficiently degassed, and water vapor was introduced to bring the pressure to 5 torr. And 13.56 MH
A chamber was placed between two electrode plates connected to a high frequency generator of Z, a plasma state was generated with an output of 25 W, and the polyester fiber was irradiated with plasma for 60 seconds. Then, a 10% aqueous solution of dimethylaminoethyl methacrylate (or a mixture of this monomer and hydroxyethyl meccrylate at a certain ratio) was added, and the mixture was heated at 50 to 60"C at 15:00 iJ1 reaction sequence 7': ,
The resulting grafted fibers were treated in a boiling water bath, dried and weighed. The grafting rate of the graph 81 was 4.5%. The grafted fibers were soaked in IN hydrochloric acid, thoroughly washed with distilled water, and invertase (Toyobo Co., Ltd.) 0,2
After soaking in a wt% aqueous solution for 24 hours, the fibers with the enzyme immobilized were obtained by washing thoroughly with water.

The enzyme-immobilized fiber was diluted with 50 ml of acetate buffer (PH 5.0) containing 5% sucrose.

The activity of the immobilized enzyme was determined by carrying out a hydrolysis reaction at 25°C for 5 minutes and measuring the amount of glucose produced using a colorimetric method. In addition, the enzyme-immobilized fiber has an acetic acid rate! The activity retention rate of Nagisa that had been exposed to 7α for 30 days was determined as follows.

Immobilized enzyme activity ---33000/g Fiber activity retention rate 90% Actual IN example 2 The grafted fibers of Example 1 were soaked in an aqueous solution of alcohol oxidase (manufactured by Toyobo) or glucose oxidase (manufactured by Toyobo) in a cool, dark place for 24 hours. Did you violate time? By thoroughly washing with water, alcohol oxidase-immobilized fibers or glucose oxidase-immobilized fibers were obtained. The thus obtained alcohol oxidase-immobilized fibers or glucose oxidase-immobilized fibers were each attached to a galvanic type oxygen electrode to form an enzyme electrode, and the responsiveness to ethanol and glucose was examined. The results are shown in the first section.
Or as shown in FIG. As is clear from FIG. 1, there is a good correlation between the degree of ethanol and the current reduction value generated at the electrode. The same applies to FIG. Also,
Table 1 shows the results of measuring 1 degree ethanol in sake during the fermentation process using this enzyme electrode. Note that Table 1 also shows the analysis results by gas chromatography.

From this, enzyme immobilization #! It can be seen that the fiber can be used as a biosensor to measure ethanol and glucose in sake.

Implementation fN3 Grafted fibers using the copolymerized monomers shown in Table 2 were produced using the same polyester fibers, equipment, and experimental conditions as in Example 1. Invertase was immobilized on the grafted fibers in the same manner as in Example 1, and the activity and activity retention of the obtained enzyme-immobilized fibers were measured, and fibers having the enzyme activities shown in Table 2 were obtained.

Example 4 Using the same equipment and experimental conditions as in Example 1, polyester film (I00 x 200 mm), polyether fiber (I00 x 200 mm), modified polyester fiber (
Cationic dyeable polyether m fiber: ], 0Ox20
0+am), dimethylaminoethyl methacrylate (R5=CH1, R2=CHs, R5
=CH1, n=2) Graft films and graft fibers were created with a 10% aqueous solution of the monomer. Invertase was immobilized on the graft film and graft fibers in the same manner as in Example 1, and the activity and activity retention of the obtained enzyme-immobilized film and fibers were measured. Fibers were obtained.

Table 1 Table 2 Table 3 Table work ≠ Luano ν call J/jl ()”/zu)th
/ ) Δ Figure 2 Procedural Amendment September 10, 1988 Patent Application No. 87998 of 1988 2, Name of Invention: Sensitive fibers, film-like carrier for immobilized enzymes 3,! 4. Those who do the right thing

Claims (1)

[Claims] 1. A fiber or film-like immobilized enzyme characterized by irradiating a fiber or film-like material with low-temperature plasma, grafting a copolymer monomer onto the surface of the material, and then immobilizing the enzyme by an ionic bonding method. Carrier for use. 2. The carrier according to claim 1, wherein the fiber or film material is organic or inorganic. 3. The copolymerizable monomer has the following chemical formula (I) or (II)
The carrier according to claim 1, which is represented by: ▲There are mathematical formulas, chemical formulas, tables, etc.▼(I) ▲There are mathematical formulas, chemical formulas, tables, etc.▼(II) (Here, R_1, R_2, R_3, R_4 are hydrogen atoms or lower alkyl groups, and n is a positive number. 4. The carrier according to claim 1, wherein the low-temperature plasma irradiation is a low-temperature plasma treatment of an inorganic gas at a gas pressure of 0.01 to 10 torr.